Vascular smooth muscle cell proliferation in response to injury is an important etiologic factor in vascular proliferative disorders such as atherosclerosis and restenosis after vascular injury caused by invasive medical techniques. Vascular injury caused by the percutaneous revascularization and other interventions stimulates the proliferation and migration of VSMC (Clowes et al., 1983, Schwartz et al., 1993, and Gordon et al., 1990). Migration of VSMC to the lumen of the injured site has been shown to be more critical to the pathogenesis of restenosis in some animal models than proliferation of VSMC per se (Schwartz et al., 1995, Schwartz et al., 1996).
This intimal accumulation of VSMC, through proliferation and migration from the media of the vessel, significantly contributes to restenosis after percutaneous revascularization interventions (Clowes et al., 1983, Schwartz et al., 1993, and Schwartz et al., 1995). The accumulation of intimal smooth muscle cell is also prominent after carotid balloon injury in rats (Clowes et al., 1983), after coronary balloon angioplasty in pigs (Schwartz et al., 1993) and in instances of restenosis after arterial dilatation in humans (Gordon et al., 1990, O'Brien et al., 1993). VSMC also contribute to the production of extracellular matrix, which increases the bulk of the neointimal mass obstructing the vessel lumen after balloon angioplasty, stenting, or other interventions that have transiently restored blood flow (Clowes et al., 1983, Schwartz et al., 1993, Schwartz et al., 1995, and Schwartz 1996).
One strategy employed for maintaining vascular patentcy after vascular injury is to induce apoptosis (cell death) of VSMC as a result of gene transfer. For example, the transfer of a replication-defective adenovirus encoding a non-phosphorylatable, constitutively active form of the retinoblastoma gene product (pRb) into VSMC inhibits the cells entry into S-phase after endovascular balloon angioplasty in rat carotid and porcine femoral artery models of restenosis (Chang et al., 1995). Adenoviral vectors encoding the herpes virus thymidine kinase (tk) gene have been introduced into porcine arteries injured with a balloon catheter (Ohno et al., 1995). When the tk gene was activated by ganciclovir treatment, intimal hyperplasia decreased.
Walsh et al. reported the prevention of restenosis with the transfer of the Fas-ligand (FasL) gene into balloon catheter injured rat carotid arteries (Sata et al., 1998). When the FasL binds to Fas (CD95) a transduction of a cytolytic signal occurs in the cell, which leads to apoptosis (Griffith, T. S. et al., 1995). Another report by Pollman and associates describes regression of vascular lesions by induction of cell death through inhibition of the death repressor gene, bc1-2 (Pollman et al., 1998). Yonemitsu et al., have described the gene transfer of p53, which was reported to prevent restenosis in balloon catheter injured rat carotid arteries (Yonemitsu et al., 1998). However, p53 induced growth arrest also occurs via the induction of p21 and, thus the p53, is not a pure "killer" gene.
The E2F-1 family of transcription factors appears to play a critical role in the transcription of certain genes required for cell cycle progression from G.sub.1 to S phase. E2F-1, the first cloned member of this family, is regulated during the cell cycle at the mRNA level by changes in transcription of the E2F-1 gene and at the protein level by complex formation with proteins such as the retinoblastoma gene product (pRb), cyclin A, and DP1. The E2F-1 gene encodes a nuclear protein, retinoblastoma-associated protein 1 ("E2F-1" or "RBAP-1"), that binds to the underphosphorylated form of human retinoblastoma (pRB), a protein that is known to repress the progression of cells towards S phase.
pRb has two known major functions. One of its functions is to sequester or inactivate the transcription factor E2F-1 which is required for activation of S phase genes. The second major function is to regulate the activity of polymerase I and III (pol I and pol III). The pRB appears to be the major player in a regulatory circuit in the late G.sub.1 phase, the so called restriction point. Moreover, pRb is involved in regulating an elusive switch point between cell cycle, differentiation and apoptosis.
A prerequisite for the growth-suppressing function of pRB is binding to the E2F-1 transcription factor, thus inhibiting transcriptional activation of genes by the E2F-1 protein which are required for DNA synthesis (Helin et al., 1992 and Nevins 1992) and cell cycle progression from G1 to S phase. Inactivation of pRb by either phosphorylation, mutation or oncoprotein binding disrupts the Rb/E2F complex and results in E2F-1 activation. Analogous, overexpression of E2F-1 can override the pRb-mediated Glarrest (Zhu et al., 1993, Qin et al., 1995, Neuman et al., 1996) and lead to either cellular transformation (Singh et al., 1994, Xu et al., 1995, Johnson et al., 1994b) or promote premature S phase entry (Qin et al., 1994 Shan and Lee 1994, Shan et al., 1996, Kowalik et al., 1995).
Several laboratories have shown a direct relationship in the transfer of the E2F-1 gene into cancer and immortalized cells and the subsequent apoptotic death of those cells. An adenovirus carrying E2F-1 (Ad.E2F-1) has been described by DeGregori et al., who observed a promotion of quiescent transformed immortalized rat fibroblast cell line, REF52, into S-phase and apoptosis (cell death), after E2F-1 gene transfer (DeGregori et al., 1997). Hunt et al., have shown that Ad.E2F-1 can kill in vitro human breast and ovarian carcinoma cell lines (Hunt et al., 1997). The E2F-1 gene, transferred by Ad.E2F-1, induces apoptosis in tumors (gliomas) in vivo, resulting in the regression of tumors, thus showing the potential therapeutic promise of E2F-1 gene transfer in cancer (Fueyo et al., 1998). Agah and associates transferred the E2F-1 gene into adult rat ventricular myocytes both in vivo and in vitro in an effort to induce myocyte proliferation after infarction (Agah et al., 1997). Instead they found that E2F-1 gene transfer led to apoptosis of the myocytes independent of the tumor suppressor protein p53.
Other reports suggested that overexpression of E2F-1 was associated with accelerated proliferation of cultured fibroblasts (Johnson et al., 1994). E2F-1 appears to have divergent growth regulatory functions, dependent on tissue type, developmental stage, and the coexistence of other genes. Prior to the present invention, no previous investigation of E2F-1 gene transfer to non-tumoral or non-immortal cell lines or tissue had been performed.
Prior to the invention herein, it was not known whether the migration and proliferation of normal vascular smooth muscle cells could be effectively reduced at the site of vascular injury by the local in vivo transformation of VSMC following injury. Moreover, it was not known whether gene therapy of VSMC could reduce atheroma formation in atherosclerotic disease states.